Many vertebrates, including man, lack the ability to manufacture a number of amino acids and therefore require these amino acids preformed in their diet. These are called essential amino acids. Plants are able to synthesize all twenty amino acids and serve as the ultimate source of the essential amino acids for humans and animals. Thus, the ability to manipulate the production and accumulation of the essential amino acids in plants is of considerable importance and value. Furthermore, the inability of animals to synthesize these amino acids provides a useful distinction between animal and plant cellular metabolism. This can be exploited for the discovery of herbicidal chemical compounds that target enzymes in the plant biosynthetic pathways of the essential amino acids and thus have low toxicity to animals.
Tryptophan is an essential amino acid. In plants, the biosynthesis of tryptophan from chorismic acid (see FIGS. 1A and 1B) requires five enzymatic steps catalyzed by anthranilate synthase (EC 4.1.3.27), anthranilate phosphoribosyl-transferase (EC 2.4.2.18), phosphoribosylanthranilate isomerase (EC 5.3.1.24), indole-3-glycerol phosphate synthase (EC 4.1.1.48) and tryptophan synthase (EC 4.2.1.20). The tryptophan pathway leads to the biosynthesis of many secondary metabolites including the hormone indole-3-acetic acid, antimicrobial phytoalexins, alkaloids and glucosinolates. Anthranilate phosphoribosyl-transferase is encoded by the PAT1 locus in Arabidopsis thaliana and the trpD locus in bacteria. Anthranilate phosphoribosyltransferase catalyzes the second step in tryptophan biosynthesis from chorismate forming 5-phosphoribosylanthranilate from anthranilate. Arabidopsis mutants in this gene are blue fluorescent under UV light due to accumulation of anthranilate compounds. Analysis of Arabidopsis plants expressing translational fusions of betaglucuronidase and different sections of the PAT1 gene indicates that the entire plastid transit peptide and the first two introns of PAT 1 are required for efficient transcription and translation (Rose, A. B. and Last, R. L. (1997) Plant J 11:455–464). Anthranilate phosphoribosyltransferase purifies from Saccharomyces cerevisiae as a dimer (Hommel, U. et al. (1989) Eur J Biochem 180:33–40).
Phosphoribosylanthranilate isomerase catalyzes the third step in tryptophan biosynthesis from chorismate forming 1-(O-carboxyphenylamino)-1-deoxyribulose-5-phosphate. Three nonallelic genes encode phosphoribosylanthranilate isomerase in Arabidopsis thaliana. All three alleles contain a plastid transit peptide at their N-terminus, are over 90% identical and are flanked by nearly identical 350 nucleotide repeats (Li, J. Y. et al. (1995) Plant Cell 7:47–461).
Indole-3-glycerol phosphate synthase catalyzes the fifth step in tryptophan biosynthesis from chorismate producing indole-glycerol phosphate from 1-(2-carboxyphenylamino)-1-deoxyribulose 5′-phosphate. Mutation of seven invariant polar residues in the active site of the enzyme from Escherichia coli have allowed the identification of catalytically essential residues. Random saturation mutagenesis indicates that K114, E163, E53 and N184 are located in the active site of the enzyme (Darimont, B. et al. (1998) Protein Sci 7:1221–1232).
Few of the genes encoding enzymes from the tryptophan pathway in corn, soybeans, rice and wheat, have been isolated and sequenced. For example, no corn, soybean, rice or wheat genes have been reported for anthranilate phosphoribosyltransferase, phosphoribosylanthranilate isomerase or indole-3-glycerol phosphate synthase. Accordingly, the availability of nucleic acid sequences encoding all or a portion of these enzymes would facilitate studies to better understand cellular biosynthetic pathways, provide genetic tools for the manipulation of those pathways, provide a means to evaluate chemical compounds for their ability to inhibit the activity of enzymes in the tryptophan biosynthetic pathway.